Gene targeting via homologous recombination in pluripotent embryonic stem (ES) cells to create chimeric animals is a powerful means of introducing specific mutations into the animal's genome. This technique also provides an opportunity to elucidate the function of genes.
The predominant method for the production of ES-cell-embryo chimeras has been the microinjection of a small number of ES cells into the blastocoel cavity followed by transfer of the developing embryo into pseudopregnant recipients for gestation. The target vector is introduced into ES cells by electropotation and the homologous recombinant clones are selected by treatment with G418 and gancyclovir. These cells are then injected into blastocysts to generate chimeric animals which are mated with the wild type animals to obtain germ line transmission of the mutation. While this method has been effectively used to produce chimeras, the cost of the necessary equipment and the length of time and level of skill required have posed practical limitations for this method.
Recently, alternative procedures to microinjection have been described based on the observation the ES cells readily aggregate with morulae. Therefore, chimeras can be produced simply by bringing the two cell populations into contact. Wood et al. (Proc. Natl. Acad. Sci. U.S.A. (1993) 90:4582-4585) described a procedure in which the embryo at the 4 to 8 cell stage or at the compact morula stage is cultured with the targeted ES cells. The ES cells are internalized into the developing blastocyst resulting in a high degree of chimerism in the animals born from these embryos. The chimeras are then cultured overnight to the blastocyte stage and transferred into pseudopregnant recipients for gestation.
A similar procedure was described by Nagy et al. (Proc. Natl. Acad. Sci. U.S.A. (1993) 90:8424-8428) involving the preparation of a "sandwich" consisting of a tetraploid embryo and a clump of ES cells.
The techniques described by Wood et al. and Nagy et al. involve culturing of embryos on a bed of ES cells to allow an unpredictable number of cells to attach to the embryo followed by the transferring of the embryo to another plate for culturing to the blastocyst stage. These aggregation methods, as compared to microinjection, require neither expensive equipment nor as high a level of expertise. However, these procedures also have limitations. First, the cells which adhere to the embryo also adhere to each other and to the pipette, and the denuded embryos also attach to each other as well as the pipette, thus causing damage to the embryo during transfer. Second, the transfer step sometimes leads to the dissociation of cells from the embryo resulting in the failure to obtain chimeric animals and damage to the embryo. Third, the number of cells attached to embryo cannot be controlled. Fourth, the contact between the cells and embryo is not very effective. In addition, the method described by Nagy et al. (1993) requires specialized skill in the preparation of the sandwich between the tetraploid embryo and the cell clumps. The number of cells in the clumps is difficult to control. Therefore, the developing embryo may be completely derived from the ES cells resulting in poor viability of the animals. A new apparatus and method of using this apparatus have now been developed which overcome the limitations of the discussed aggregation methods. The apparatus and method provide for: 1) minimal handling of denuded embryos which results in high rate of blastulation; 2) effective control of the number of cells to be incorporated into the embryos; 3) monitoring of incorporation of ES cells into the embryos; and 4) no opportunity for embryos to adhere to each other. In addition, no special skills are required to use the apparatus and only one step of co-culture is involved.